Lov-D acyltransferase mediated acylation

Abstract

Methods for the improved acylation of chemical substrates using LovD acyltransferases, thioesters having acyl groups, and (i) thiol scavengers and/or (ii) precipitating agents are presented. An improved method for the production of simvastatin using (i) activated charcoal as a thiol scavenger and/or (ii) ammonium hydroxide as a precipitating agent is also presented.

What is claimed is: 
     
       1. A method of making a statin compound, comprising contacting a LovD acyltransferase substrate with a LovD acyltransferase, wherein said LovD acyltransferase is a recombinant variant comprising SEQ ID NO:16, in the presence of a thioester donor and an agent selected from a thiol scavenger, a precipitating agent and combinations thereof, under conditions which yield a statin compound. 
     
     
       2. The method of  claim 1  in which the LovD acyltransferase substrate is monacolin J. 
     
     
       3. The method of  claim 2  in which the monacolin J is a monacolin J hydroxy acid salt. 
     
     
       4. The method of  claim 2  in which the monacolin J is monacolin J lactone. 
     
     
       5. The method of  claim 1  in which the LovD acyltransferase substrate is lovastatin. 
     
     
       6. The method of  claim 5  in which the lovastatin is a lovastatin hydroxy acid salt. 
     
     
       7. The method of  claim 5  in which the lovastatin is lovastatin lactone. 
     
     
       8. The method of  claim 1  in which the LovD acyltransferase substrate is 6-hydroxy-6-des-methyl-monacolin-J. 
     
     
       9. The method of  claim 8  in which the 6-hydroxyl-6-desmethylmonacolin J is a 6-hydroxy-6-des-methyl monacolin J hydroxy acid salt. 
     
     
       10. The method of  claim 8  in which the 6-hydroxy-6-desmethyl monacolin J is 6-hydroxyl-6-des-methyl monacolin J lactone. 
     
     
       11. The method of  claim 1  in which the LovD acyltransferase substrate is pravastatin. 
     
     
       12. The method of  claim 11  in which the pravastatin is a pravastatin hydroxy acid salt. 
     
     
       13. The method of  claim 11  in which the pravastatin is a pravastatin lactone. 
     
     
       14. The method of  claim 1 , in which the thioester donor is an alpha-dimethylbutyryl thioester. 
     
     
       15. The method of  claim 14  in which the alpha-dimethylbutyryl thioester is selected from the group consisting of α-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP), dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP), dimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG), dimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB), S-2-acetamidoethyl 2,2-dimethylbutanethioate, S-acetamidomethyl 2,2-dimethylbutanethioate and methyl 2-(2,2-dimethylbutanoylthio)acetate. 
     
     
       16. The method of  claim 15  in which the alpha-dimethylbutyryl thioester is DMB-S-MMP. 
     
     
       17. The method of  claim 14 , which is carried out in an aqueous medium at a pH in the range of about pH 8 to pH 9.5. 
     
     
       18. The method of  claim 14 , which is carried out in an aqueous buffer having an initial pH of about pH 9. 
     
     
       19. The method of  claim 14  in which the agent is a thiol scavenging agent. 
     
     
       20. The method of  claim 19  in which the thiol scavenging agent is activated charcoal. 
     
     
       21. The method of  claim 14  in which the agent is a precipitating agent. 
     
     
       22. The method of  claim 21  in which the precipitating agent is ammonium hydroxide. 
     
     
       23. A method of making simvastatin hydroxy acid ammonium salt comprising contacting monacolin J or lovastatin with a LovD acyltransferase, wherein said LovD acyltransferase is a recombinant variant comprising SEQ ID NO:16, in the presence of an alpha-dimethylbutyryl thioester donor and ammonium hydroxide under conditions which yield simvastatin hydroxy acid ammonium salt. 
     
     
       24. The method of  claim 23 , which is carried out in aqueous solution at a pH in the range of about pH 8 to pH 9.5. 
     
     
       25. The method of  claim 23 , which is carried out in an aqueous buffer having an initial pH of about pH 9.